With the advent of real-time (kinetic) polymerase chain reaction (PCR) analysis, using appropriate standards and sample preparation techniques it is now possible to directly identify the presence of specific nucleic acid sequences and even quantify the number of target nucleic acid molecules in a sample without the need for post-PCR analysis methods, such as gel electrophoresis. Such technology may be useful for identification and/or quantification of specific organisms (e.g. bacteria or other pathogens), identification of specific genes or transcripts, viral detection and quantification, as well as a myriad of other applications. However, one of the risks associated with testing samples by PCR is the occurrence of false negatives.
While a positive and negative control are normally run for every PCR master mix to ensure the integrity of the reagents, inhibition of the PCR by the sample matrix may cause an individual test sample to report a negative result, even if there is target template present in the reaction. In quantitative real-time PCR this is even more of a concern, as partial PCR inhibition may lead to inaccurate quantification results. Therefore, it is desirable to include an internal positive control in each individual reaction to prevent the reporting of false negatives and to potentially allow accurate adjustments to quantitative data.
At present, only a few internal positive control reagents are available commercially. For example, an internal control is available from Applied Biosystems as a component that can be incorporated into TaqMan® (Applied Biosystems) PCR kits and can be spiked into samples to distinguish true target negatives from negatives due to PCR inhibition. The TaqMan® internal positive control sequence can distinguish different types of negative results: (a) a negative call for the target sequence and a positive call for the IPC suggests that no target sequence is present; and (b) a negative call for the target sequence and negative call for the IPC suggests PCR inhibition.
Another example of an internal positive control was reported by Rosenstraus M., et al., (1998) J. Clinical Microbiol 36(1):191-197. That group constructed internal control nucleic acids for use in COBAS AMPLICOR tests for Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and human hepatitis C Virus. The internal control sequence constructed by Rosenstraus et al. had primer binding regions identical to those of the target sequence primers but included a unique probe binding region that differentiated the internal control sequence from amplified target nucleic acid. Because only 20 copies of the internal control sequence were introduced into each test sample, a positive internal control signal indicated that amplification was sufficient to generate a positive signal from targets present at the limit of test sensitivity.
Using the Rosenstraus internal control sequence as a model, Danbing Ke, et al., (2000) Clinical Chemistry 46(3): 324-331, constructed an internal control sequence for a PCR assay used to quantify Group B Streptococci. However, this internal control had to be constructed de novo to make it compatible with the Streptococci Group B assay. Using the method of Rosenstraus it was necessary to synthesize the internal control to utilize the same primer binding sequence set as the Streptococci Group B assay used.
A similar limitation is demonstrated in Wellinghausen et al., (2001) Appl Environ Microbiol. 67(9):3985-93, which used an internal control that utilizes a known (naturally occurring) lambda phage DNA sequence flanked by assay gene-specific primer regions. This internal control is also designed only for use in a particular assay.
A recent abstract from Hartman et al. (Hartman et al., ASM General Meeting Abstracts, May 2003) also includes similar limitations. Hartman et al. disclosed a Taqman® internal control that has mutated primers and probe binding sites. The assay is designed to amplify the same target DNA, but with different primers and probes. This assay is also specific to the Taqman® assay and would not be universally applicable.
As shown, known methods of providing internal controls have limitations due in large part to the need for sequence customization and reaction specificity. Therefore, there remains a need for an internal control molecule designed as part of a comprehensive internal control system and methods for its use that can be universally incorporated into virtually any PCR assay.
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